Association of killer cell immunoglobulin-like receptors and their cognate HLA class I ligands with susceptibility to acute myeloid leukemia in Iranian patients

Acute myeloid leukemia (AML) is one of the most prevalent leukemia in adults. Among the various NK receptors, killer immunoglobulin-like receptors (KIRs) carry out indispensable roles in NK cell development and function through engaging with class I human leukocyte antigens (HLA-I) as their ligands. Besides divergent KIR and HLA loci, KIR/HLA-I combinations have a significant effect on NK cell response. In this case–control study, we aimed to verify the association of KIR/HLA-I combinations with susceptibility to AML in the Southwestern Iranian population. KIR and HLA genotyping was performed with PCR-SSP by some novel primers for 181 patients with AML and 181 healthy controls. According to our results, the frequencies of KIR3DS1 (p = 0.0001, OR = 2.32, 95% CI 1.51–3.58), KIR2DS4fl (p = 0.02, OR = 1.53, 95% CI 1.05–2.21), CxT4 genotypes (p = 0.03, OR = 2.0, 95% CI 1.05–3.82), and T4 gene cluster (p = 0.01, OR = 1.99, 95% CI 1.17–3.41) were significantly higher in patients than controls, while C1/C2 genotype (p = 0.00002, OR = 0.39, 95% CI 0.25–0.61), HLA-A Bw4 (p = 0.02, OR = 0.6, 95% CI 0.38–0.94), and HLA-A*11 (p = 0.03, OR = 0.57, 95% CI 0.34–0.95) alleles were more frequent in controls. In addition, inhibitory (i)KIR/HLA-I combinations analysis revealed higher frequencies of KIR2DL1( +)/HLA-C2( +), KIR2DL2/3( +)/HLA-C1( +), KIR3DL1( +)/HLA-A Bw4( +), and KIR3DL2( +)/HLA-A*03/11( +) in the control group (p = 0.002, OR = 0.49, 95% CI 0.3–0.78; p = 0.04, OR = 0.62, 95% CI 0.39–0.99; p = 0.04, OR = 0.63, 95% CI 0.4–0.99; and p = 0.03, OR = 0.62, 95% CI 0.4–0.95, respectively). Overall, the number of iKIR/HLA-I combinations was more in the control group. Moreover, KIR3DS1( +)/HLA-B Bw4Ile80( +) and the sum of HLA-B Bw4/A Bw4 combined with KIR3DS1 as activating KIR/HLA-I combinations were more frequent among patients than controls (p = 0.01, OR = 1.99, 95% CI 1.14–3.49 and p = 0.005, OR = 1.97, 95% CI 1.22–3.19, respectively). In conclusion, our results postulate that inhibitory combinations play a protective role against AML by developing potent NK cells during education. It is noteworthy that KIR/HLA-I combination studies can be applicable in donor selection for allogeneic NK cell therapy in hematological malignancies.


Results
To investigate the combined effect of KIR and their cognate HLA-I ligands on susceptibility to AML, KIR and HLA genotyping was applied for 181 patients, and the results were compared with 181 healthy controls. Patients with an average age of 49.94 ± 16.91 years (range 17-87) included 71 women (50.18 ± 18.1 years) and 110 men (49.79 ± 16.17 years).

Frequencies of KIR genes and genotypes in patients with AML and controls. The frequency of
KIR genes is shown in Table 1. As shown, only the frequency of KIR3DS1 was significantly higher in patients than in controls (p = 0.0001, OR = 2.32, 95% CI 1.51-3.58). KIR2DS4 fl allele and fl/fl genotype were more common in patients than controls (p = 0.02, OR = 1.53, 95% CI 1.05-2.21 and p = 0.03, OR = 2.04, 95% CI 1.03-4.07, respectively) (Supplementary Table S1). Our data showed no significant difference in AA and Bx genotypes between patients and controls; however, the CxT4 genotype and T4 gene cluster were more frequent in patients compared to controls (p = 0.03, OR = 2, 95% CI 1.05-3.82 and p = 0.01, OR = 1.99, 95% CI 1.17-3.41, respectively) ( Table 2). In total, a higher frequency of aKIRs ≥ 4 was detected in the patient group than in the control group, although this difference was not significant.
KIR profiles of patients and healthy individuals are shown in Supplementary Table S2. We found 93 different KIR gene profiles with known patterns, of which 28 were patient-specific, 38 were control-specific, and 27 profiles were identified in both groups.
Frequencies of HLA-I ligands in patients with AML and controls. According to our results (Table 3), there was no significant difference between HLA-C allotypes (C1 and C2), HLA-B allotypes (Bw4 and Bw6), and HLA-B Bw4 isoforms (Bw4 Ile80 and Bw4 Thr80 ) between patients and controls. While our data showed a higher frequency of HLA-A Bw4 in the control group than in patients with AML (p = 0.02, OR = 0.6, 95% CI 0.38-0.94) which seems to be due to the difference of A*23/24 as a part of HLA-A Bw4. Also, HLA-A*11 was more prevalent in controls (p = 0.03, OR = 0.57, 95% CI 0.34-0.95).
The average age of patients with AML at the time of diagnosis, according to ACS and HMRN, was about 68 years. However, the median age of our patients was about 50 years, which is more comparable to Brazilian (less than 60 years) and Saudi Arabian (53 years) patients 32,33 . The age variation of patients among different populations might be due to their genetic origins and environmental factors 34,35 .
In our study, KIR3DS1 frequency was significantly higher in patients than in controls, and we suggested 3DS1 as a risk factor for AML. Misra et al. also reported a higher frequency of aKIRs, especially KIR3DS1among their patients with childhood ALL 36 . Unlike our study, Shahsavar et al. showed a lower frequency of 3DS1 in patients with AML, but it was not significant 37 . In contrast, Varbanova et al. reported a significantly lower frequency of 3DS1 in patients with AML 38 . Also, the 2DS4fl allele and fl/fl genotype were significantly increased among our patients, while Giebel et al. found a higher frequency of the 2DS4del allele in their patients with CML compared to the control group 39 . Furthermore, our results revealed that the T4 cluster (KIR3DS1-2DL5-2DS1-2DS5) and CxT4 genotype were significantly more frequent in patients than in controls. Therefore, the telomeric gene cluster with more aKIRs can be considered a risk factor for AML. These results describe an activating trend of NK response in patients with AML.     www.nature.com/scientificreports/ Moreover, we found that although there was no significant difference between patients and controls based on the frequency of HLA-C allotypes, C1/C1 and C2/C2 genotypes were more frequent in patients, and C1/C2 genotype was dominant in the control group. It seems C1/C2 genotype has heterozygote advantage and plays a protective role in the control group 40 . Indeed, the heterogenicity led to more variety of HLA-I ligands and KIR/ HLA-I combinations in the control group. Similarly, no association was found between HLA-C alleles and leukemia in Hispanic/non-Hispanic and Bulgarian patients 31,38 . However, a study in the Thai population reported the low frequency of HLA-C2 and C1/C1 genotypes in patients with AML 41 . Whereas studies in the Iranian and German populations stated contradictory results and introduced HLA-C2 as a susceptibility factor to ALL 37,42 .
In terms of HLA-B allotypes (Bw4 and Bw6), genotypes (Bw4/Bw4, Bw6/Bw6, and Bw4/Bw6) and Bw4 isoforms (Bw4 Ile80 and Bw4 Thr80 ), there was no significant difference between two groups. Unlike our results, Shahsavar et al. indicated a higher frequency of HLA-B Bw4 Ile80 in patients with AML. They also found no significant difference in HLA-A Bw4 between patients and controls 37 . Middleton et al. also observed a higher frequency of HLA-B Bw4 Ile80 in patients with AML. They also considered the Bw4/Bw4 genotype as a predisposing and the Bw6/Bw6 genotype as a protective factor in Turkish white CML patients 43 . In contrast, the study on the Bulgarian population showed a higher frequency of HLA-B Bw4 Thr80 in myeloid leukemia patients and the Bw4/Bw4 genotype in leukemia patients 38 .
To investigate KIR/HLA-I combinations, we considered different states, including (1)  We detected the same trend in the KIR2DL2/3/HLA-C1 combination. Double positives form of this combination was common among the control group, but KIR2DL2/3( +)/HLA-C1(-) revealed a higher frequency in our patient group.
Also, we identified an increased frequency of the KIR3DL1( +)/HLA-A Bw4( +) combination among the control group, but KIR3DL1( +)/HLA-A Bw4(-) was more common in the AML group. Moreover, the same trend was also found in the KIR3DL2/HLA-A*03/11 combinations. Consistent with our results, Varbanova et al. Overall, our results revealed a boost in the frequency of iKIRs and their cognate HLA-I ligands in the control group but a higher frequency of iKIRs in the absence of their ligands in AML. We also observed a higher frequency of aKIR/HLA-I combinations in the AML group and the presence of HLA-I ligands in the absence of their corresponding aKIRs in the control group. To explain these results, we propose two different models. First, based on higher frequencies of aKIRs and aKIR/HLA-I combinations in patients, it seems that their NK cells are hyperresponsive and become exhausted through challenging with malignant cells. These NK cells are not able to eradicate malignant cells, but they cause chronic inflammation in the tumor microenvironment. This can be considered a reason for the delayed onset of AML, as most initial diagnoses are made between 50 and 70 years. Second, which is more plausible, NK cells represent an essential actor in immunosurveillance. To become more efficient, these cells go through licensing process in which just those NK cells that recognize HLA-I ligands by their inhibitory receptors gain the ability to respond against cancerous cells. In this connection, our results indicated a higher frequency of iKIR/HLA-I combinations in the control group; therefore, it seems that these licensed NK cells are more eligible for early removal of cancer cells in the control group.
To make inferences, we implied a higher frequency of KIR3DS1, KIR2DS4fl, CxT4 genotypes, and T4 genes cluster beside a higher frequency of aKIRs in patients with AML. Also, our data showed higher frequencies of C1/C2 genotype, A Bw4, and A*11 alleles in the control group; therefore, we suggest these elements as a protective factor. To summarize the results of KIR/HLA-I combinations, the presence of iKIRs with their cognate ligands, such as KIR2DL1( +)/HLA-C2( +), KIR2DL2/3( +)/HLA-C1( +) and KIR3DL2( +)/HLA-A*03/11( +) were more frequent in the control group. In contrast, the presence of these iKIRs without their cognate ligands had a higher frequency in patients with AML. Furthermore, aKIR/HLA-I combinations, such as KIR3DS1( +)/HLA-B Bw4 Ile80 ( +) and KIR3DS1( +)/HLA-Bw4( +), were more prevalent in patients, while the presence of HLA-I ligands (B Bw4 Ile80 , B Bw4 Thr80 , and A Bw4) without their corresponding receptor, KIR3DS1, revealed an increased frequency in controls. In fact, regarding the lower frequency of iKIRs/HLA-I in patients, their NK cells were not properly educated and would not be sufficiently competent for inhibiting cancer cell development.
It should not be ignored that NK cell response is influenced by many different factors, such as various signals from different kinds of receptors as well as malignant cell status. Indeed, our study is a piece of the puzzle in NK  44 . Due to their low white blood cell counts and low DNA yield, patients undergoing intensive chemotherapy were omitted from the study. Demographic information was collected from patients' medical records. As the control group, DNA samples from 181 sex/ethnicity-matched healthy adults were collected from the same geographic region, Fars province in southwestern Iran.
KIR and HLA genotyping. Genomic DNA was extracted from 300 µl of whole blood samples using the non-enzymatic salting-out method, and the concentration was adjusted at 30 ng/μl 45 . KIR genes (2DL1, 2DL2, 2DL3, 2DL5, 2DS1, 2DS2, 2DS3, 2DS4, 2DS5, 3DL1 and 3DS1) were genotyped by polymerase chain reaction with sequence-specific primers (PCR-SSP) method using 11 specific primer pairs designed by Vilches et al. 46 . Moreover, an additional pair of primers based on the sequences reported by Ashuri et al. were used to discriminate fl and del variants of KIR2DS4 47 . DNA samples with known KIR patterns were gifted from the Shiraz Institute for Cancer Research as controls.
Regarding the importance of HLA-A*03 and A*11 interaction with KIR3DL2 and the lack of specific primers for detecting these HLA alleles with PCR-SSP, we designed two novel primer pairs. In addition, the previously designed HLA-A Bw4 specific primers identify not only HLA-A*23/24/32 alleles but also HLA-A*25, which has been proven not to interact with KIR3DL1 as a ligand 23,24 . Therefore we designed two specific forward primers (Table 8), which in combination with the previously published reverse primer, can specifically amplify HLA-A*23/24/32 alleles 48 . To discriminate Bw4 from Bw6-bearing allotypes and due to the lack of appropriate Bw6specific pair of primers, we optimized Bw6-specific primer pair by combining Tajik's Bw4 Ile80 forward primer with Hong's Bw6 reverse primer 48,49 . For designing the new primers, sequences of certain HLA alleles were extracted from the IPD-IMGT/HLA database (ftp:// ftp. ebi. ac. uk/ pub/ datab ases/ ipd/ imgt/ hla/). Then BioEdit version 7.2, CLC sequence viewer version 7.8.1, and AlleleID version 7.7 software were used to design and evaluate the thermodynamic properties and the possibility of forming secondary structures. Also, the Primer-BLAST was employed to check the specificity of the primers.
DNA samples with known HLA alleles -typed by high-resolution method -were included as controls from our previous study, which was performed in collaboration with the National Reference Laboratory for Histocompatibility, Geneva, Switzerland 50 .
Each PCR reaction contained a couple of specific primes and a pair of primers for amplifying a non-polymorphic fragment of the human HLA-DRα gene as the internal control. The negative control was a PCR mixture without template DNA, and DNA samples with known KIR and HLA genotypes confirmed by high-resolution methods were used as the positive control. Each PCR reaction was performed in a total volume of 10 μl containing www.nature.com/scientificreports/ 5 μl master mix (2x) including Taq DNA polymerase (Ampliqon A/S, Denmark), 0.5 μM of each specific primer, 0.1 μM of each internal control primer, and 30 ng of genomic DNA. The temperature profiles of previously designed primers were applied according to Tajik's study, and the thermal conditions of newly designed primers are summarized in Table 8. PCR products of different HLA and KIR genes were verified by agarose gel electrophoresis (2%) containing DNA-safe stain, and a 50 bp DNA ladder was loaded in every gel. To differentiate between 2DS4 full length (fl) and deleted (del) alleles, electrophoresis was done on 4% agarose gel. After KIR typing, genotypes of each sample were determined based on an allele frequency database (http:// www. allel efreq uenci es. net/ kir60 01a. asp).
Statistical analysis. The frequency of genes, genotypes, and KIR/HLA-I combinations between patients and healthy controls was compared using the Chi-square test based on a 2 × 2 contingency table. To prevent overestimation of statistical significance for small data, Yates' correction was used when at least one cell of the 2 × 2 contingency table had a count smaller than 5. Data were analyzed using SPSS 26, and p < 0.05 was considered statistically significant. The odds ratio (OR) and 95% confidence interval (CI) were calculated to investigate the significance of the association of KIR genes and their cognate HLA-I ligands with susceptibility to AML.

Data availability
The data that support the findings of this study are available from the corresponding author upon reasonable request.